2.4. Histological analysis

Positive Alizarin red staining indicates the osteogenic differentiation of GFs. The cells from the four experimental groups were cultured in a monolayer as described in the osteogenic differentiation assay. After a period of 21 days, the wells were washed with distilled water twice and fixed for 15 min at room temperature with 10% w/v neutral buffered formalin (Anachemia Canada Inc., Quebec, Canada). Alizarin red staining was used to stain the cultured wells for 10 min. The wells were washed again on a shaker for 15 min. Finally, images were captured and used to qualitatively assess the alizarin-stained mineralized nodules by light microscopy.

Safranin O was used following chondrogenic differentiation to detect if any sulphated proteoglycans are produced in the extracellular matrix. After 21 days of chondrogenic differentiation, the pellets were fixed overnight at 4 °C in 10% w/v neutral-buffered formalin. To preserve the cells and increase hydrophobicity, the pellets were dehydrated by immersing them in incrementally higher concentrations of ethanol. The pellets were then embedded in paraffin and cut into 5 μm sections. To detect whether a matrix of sulphated proteoglycans formed within the pellets, the mounted sections of the pellets were stained with 0.01% w/v safranin-O and counterstained with 0.02% w/v fast green (Sigma-Aldrich, Missouri, US). Safranin O staining was used to assess the chondrogenic differentiation of the gingival fibroblasts. Safranin O stains the acidic proteoglycans with an orange-red color. Fast green is a sulphate group which binds strongly to the amino group on protein and stains the non-collagen sites.

To identify the absence or presence of the ECM components collagen type I and collagen type II, we performed immunofluorescent analysis. 5 μm sections were deparaffinized after treatment with UltraClear™ solution followed by ethanol and distilled water. Due to the formation of methylene bridges during fixation, the slides were incubated for 30 min at room temperature in an antigen retrieval enzyme, protease XXV (AP-9006–005, Thermo Scientific, Massachusetts, US). To increase the specificity of the antibodies, the slides were incubated in hyaluronidase (H6254, Sigma-Aldrich, Missouri, US) for 30 min at 37 °C. The sections were then incubated in bovine serum albumin (BSA) 5% w/v to reduce non-specific binding of the antibodies. After BSA incubation, the pellet slices were incubated in primary antibodies: rabbit anti-collagen I (CL50111AP-1, Cedarlane, Ontario, Canada) and mouse anti-collagen II (II-II6B3, Developmental Studies Hybridoma Bank, Iowa, US) overnight at 4 °C with a 1:200 dilution. The preceding step was followed by incubation with a fluorescently-conjugated secondary antibody at a dilution of 1:200 with goat anti-rabbit IgG (H&L Alexa Fluor 594, Abcam, UK) for collagen type I and goat anti-mouse IgG (H&L Alexa Fluor 488, Abcam, UK) for collagen type II. The sections were stained with DAPI (4′, 6-diamidino-2-phenylindole, Cedarlane) to stain the cell nuclei, and mounted with a 1:1 glycerol-PBS solution. Immunofluorescent images were visualized by an Eclipse Ti-S microscope (Nikon Canada, Ontario, Canada).